Development of Green HPLC-PDA Method for Quantification and Extraction Optimization of Dihydromyricetin, Vitexin, and Isovitexin in the LCD20 Herbal Remedy

Dihydromyricetin, vitexin, and isovitexin are bioactive compounds in the LCD20 herbal remedy, valued for their role in peptic ulcer treatment. This study presents the development and validation of a green HPLC-PDA method for the simultaneous quantification of these three compounds in the remedy. Chromatographic separation was achieved using an InertSustain C18 column (4.6 × 250 mm, 5 μm) with a mobile phase of acetonitrile and 0.1% formic acid (13:87, v/v) and detection at 330 nm. The method demonstrated excellent linearity with ranges of 25–1000 μg/mL for dihydromyricetin and 5–200 μg/mL for vitexin and isovitexin. The method demonstrated high sensitivity with low limits of detection and quantification. The method also exhibited satisfactory specificity, accuracy, precision, sensitivity, and stability. Greenness assessment confirmed the method’s alignment with green analytical chemistry, with AGREE (0.56), Eco-Scale (77), ComplexGAPI classification as low risk, and a BAGI score of 67.5. Finally, the validated method was utilized to optimize the manufacturing process of the herbal extract via response surface methodology; conditions of 84.3% ethanol, 11.4 mL/g solvent-to-solid ratio, and 4.86 h were established for maximum efficiency. This approach ensures reliable quantification and extraction, aiding the standardization of LCD20 for pharmaceutical use.